Epidemics and outbreaks of dengue caused by one or more of the four dengue virus serotypes continue to pose a major public health problem in most tropical and subtropical areas. A safe and effective vaccine against dengue is currently not available. Recently, a DEN4 deletion mutant that had been constructed and characterized in this laboratory was evaluated for safety and immunogenicity in human volunteers. Results from a total of 20 healthy adults indicated that this live dengue type 4 virus vaccine was well tolerated and did not cause serious illness. After a single dose all volunteers sero-converted with a 7-fold or greater rise in serum neutralizing antibody titer. However, a mild asymptomatic macular rash developed in 10 volunteers and a transient elevation in serum liver enzyme (ALT) level was found in 5 volunteers. This indicates that this live vaccine candidate is not sufficiently attenuated. Passive immunization using dengue virus neutralizing antibodies provides an attractive alternative to vaccine for prevention of dengue virus infection. Toward this goal, we initiated a new project to identify and characterize antibodies of chimpanzees or humans that effectively neutralize each of the four dengue virus serotypes. We employed the technique of phage display of combinatorial antibody, which has provided a powerful tool for isolation of human or chimpanzee antibodies to important viral pathogens. A combinatorial antibody library was constructed from the bone marrow lymphocytes of two chimpanzees that had been infected following intrahepatic inoculation with DEN4 RNA. Panning of the phage library and subsequent screening have allowed identification of four distinct groups of Fab antibodies reactive to DEN4 on the basis of their sequences in the heavy chain variable region. As predicted, a computer search revealed that these chimpanzee Fabs shared a high sequence homology with the human immunoglobulin IgG homologs. Three of these Fabs co-precipitated DEN4 PrM and E, whereas the fourth Fab precipitated PrM alone. Competitive ELISA showed that two of these Fabs reacted with overlapping sites on E, whereas the other two Fabs reacted with overlapping sites on PrM. Each of these Fab clones weakly neutralized DEN4 in vitro only in the presence of an anti-Fab antibody, suggesting that a multivalent form of these antibodies was required for the neutralizing activity. High level expression of the full immunoglobulin IgG molecules in mammalian cells should allow determination if any of the antibodies from the Fab clones is capable of neutralizing DEN4. These are the first monoclonal antibodies specific to DEN4 from higher primates (chimpanzees). To expand the antibody repertoire, another combinatorial antibody library was successfully constructed from chimpanzees hyper-immunized with each of the four dengue viruses. This new antibody library should prove valuable for identification of neutralizing antibodies against each of the four dengue viruses for use in immunoprophylaxis.